Method of treating vitamin B12 deficiency

ABSTRACT

A novel method and composition for treating vitamin B 12  deficiency mammals that fail to respond to oral vitamin B 12  therapy.

CROSS-REFERENCE TO PRIOR APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/262,677, filed Oct. 31, 2008, now U.S. Pat. No. 8,022,048, whichclaims the benefit of U.S. Provisional Application No. 60/984,898, filedNov. 2, 2007; U.S. Provisional Application No. 61/020,108, filed Jan. 9,2008; and U.S. Provisional Application No. 61/083,566, filed Jul. 25,2008, each of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to methods of treating vitaminB₁₂ deficiency and pharmaceutical compositions for such treatment.

BACKGROUND OF THE INVENTION

Vitamin B₁₂ is important for the normal functioning of the brain andnervous system and for the formation of blood. It is involved in themetabolism of every cell of the body, especially affecting the DNAsynthesis and regulation but also fatty acid synthesis and energyproduction. Its effects are still not completely known.

Cyanocobalamin is the most stable and widely used form of vitamin B₁₂.It is bound to plasma proteins and stored in the liver. Vitamin B₁₂ isexcreted in the bile and undergoes some enterohepatic recycling.Absorbed vitamin B₁₂ is transported via specific B₁₂ binding proteins,transcobalamin I and II, to the various tissues. The liver is the mainorgan for vitamin B₁₂ storage.

Vitamin B₁₂ deficiency can potentially cause severe and irreversibledamage, especially to the brain and nervous system. Oral tabletscontaining vitamin B₁₂ have been developed to treat vitamin B₁₂deficiency. However, many patients with vitamin B₁₂ deficiency do notrespond to oral vitamin B₁₂ treatment. There is a need to develop atreatment for these patients.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention is directed to a method for treating vitaminB₁₂ deficiency in a subject, comprising the steps of (a) preparing apharmaceutical composition for oral administration containing (1)vitamin B₁₂ and (2) at least one substance selected from the groupconsisting of N-[8-(2-hydroxybenzoyl)amino]caprylic acid and itspharmaceutically acceptable salts; and (b) administering thepharmaceutical composition to the subject to effectively treat saidvitamin B₁₂ deficiency.

Another aspect of the invention is directed to a pharmaceuticalcomposition for treating vitamin B₁₂ deficiency in a subject, comprising(1) vitamin B₁₂ and (2) at least one substance selected from the groupconsisting of N-[8-(2-hydroxybenzoyl)amino]caprylic acid and itspharmaceutically acceptable salts; wherein said subject had failed torespond to existing oral vitamin B₁₂ treatment.

The contents of the patents and publications cited herein and thecontents of these documents cited in these patents and publications arehereby incorporated herein by reference to the extent permitted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of serum vitamin B₁₂ concentration as a function oftime.

DETAILED DESCRIPTION

As used herein, the term “SNAC” meansSodium-N-salicyloyl-8-aminocaprylate, Monosodium 8-(N-salicyloylamino)octanoate, N-(salicyloyl)-8-aminooctanoic acid monosodium salt,monosodium N-{8-(2phenoxybenzoyl)amino}octanoate, E414 monosodium saltor sodium 8-[(2-hydroxybenzoyl)amino]octanoate. It has the structure

“N-[8-(2-hydroxybenzoyl)amino]caprylic acid” has an empirical formulaC₁₅H₂₁NO₄.

The term “Vitamin B₁₂” means any member of a group of cobalt-containingcompounds known as cobalamins which include, but is not limited tocyanocobalamin, hydroxocobalamin, methylcobalamin, and5-deoxyadenosylcobalamin.

The term “treatment” or “treating” means any treatment of a disease ordisorder in a mammal, including: preventing or protecting against thedisease or disorder, that is, causing the clinical symptoms not todevelop; inhibiting the disease or disorder, that is, arresting orsuppressing the development of clinical symptoms; and/or relieving thedisease or disorder, that is, causing the regression of clinicalsymptoms. The term “mammal” include human subjects.

The terms “carrier, excipient, emulsifier, stabilizer, sweetener,flavoring agent, diluent, coloring agent, solubilizing agent” are asdefined in the Handbook of Pharmaceutical Excipients (fourth edition) byRaymond C. Rowe, Paul J. Sheskey and Paul J. Weller, the content ofwhich is herein incorporated by reference.

The term “intrinsic factor protein” means is a glycoprotein produced bythe parietal cells of the stomach. It is necessary for the absorption ofvitamin B₁₂ later on in the terminal ileum.

In a preferred embodiment, the treatment is directed to subjects thathad failed to respond to existing oral vitamin B₁₂ treatment.Preferably, tablets are used for the treatment. Such tablets containfrom about 0.01 mg to about 25 mg of vitamin B₁₂ and from about 1 mg toabout 600 mg of SNAC each, preferably from about 0.02 mg to about 25 mgof vitamin B₁₂ and more preferably from about 0.1 mg to about 20 mg ofvitamin B₁₂ and the most preferably from about 0.5 mg to 10 mg ofvitamin B₁₂ and from about 10 mg to about 200 mg of SNAC in each tablet.

The preferred weight ratio of vitamin B₁₂ and SNAC in the tablet is fromabout 2:1 to about 1:700, more preferably from about 1:2 to about 1:600or from about 1:3 to about 1:20 and the most preferably from about 1:4to about 1:10.

In a preferred embodiment, the pharmaceutical composition is in the formof tablets. Preferrably, each tablet contains from about 0.01 mg toabout 25 mg of vitamin B₁₂ and from about 50 mg to about 600 mg of SNAC.More preferably, each tablet contains from about 0.02 mg to about 20 mgof vitamin B₁₂. More preferably, each tablet contains from about 0.1 mgto about 10 mg of vitamin B₁₂. The most preferably, each tablet containsabout 15 to 20 mg of vitamin B₁₂ and about 50 to 100 mg of SNAC, orabout 0.1 to 1.5 mg of vitamin B₁₂ and about 25 to 150 mg of SNAC.

In another preferred embodiment, the tablet further contains at leastone of a carrier, excipient, emulsifier, stabilizer, sweetener,flavoring agent, diluent, coloring agent, solubilizing agent orcombinations thereof.

In another preferred embodiment, the tablet optionally contains fromabout 1 to 25 mg of Capmul PG-8 and optionally contains from about 0.5to 10 mg of providone. Preferably, Capmul PG-8 is in an amount fromabout 2 to 20 mg and Providone is in an amount from about 1 to 8 mg.Preferably, Capmul PG-8 is in an amount from about 5 to 15 mg and theProvidone is in an amount from about 1.5 to 5 mg. More preferably,Capmul PG-8 is in an amount from about 5 to 10 mg and Providone is in anamount from about 1.5 to 5 mg.

Without intending to be bound by any particular theory of operation, itis believed that gastrointestinal absorption of vitamin B₁₂ depends onthe presence of sufficient intrinsic factor protein, secreted fromgastric parietal cells. The average diet supplies about 10 mcg/day ofvitamin B₁₂ in a protein-bound form that is available for absorptionafter normal digestion. Vitamin B₁₂ is bound to intrinsic factor duringtransit through the stomach; separation occurs in the terminal ileum,and vitamin B₁₂ enters the mucosal cell for absorption via a receptormediated process. It is then transported by the transcobalamin bindingproteins. A small amount (approximately 1% of the total amount ingested)is absorbed by simple diffusion, but this mechanism is adequate onlywith very large doses. It is also believed that SNAC will allow B₁₂ tobypass its usual receptor mediated process.

The following examples are given as specific illustrations of theinvention. It should be understood, however, that the invention is notlimited to the specific details set forth in the examples. All parts andpercentages in the examples, as well as in the remainder of thespecification, are by weight unless otherwise specified.

Further, any range of numbers recited in the specification or paragraphshereinafter describing or claiming various aspects of the invention,such as that representing a particular set of properties, units ofmeasure, conditions, physical states or percentages, is intended toliterally incorporate expressly herein by reference or otherwise, anynumber falling within such range, including any subset of numbers orranges subsumed within any range so recited. The term “about” when usedas a modifier for, or in conjunction with, a variable, is intended toconvey that the numbers and ranges disclosed herein are flexible andthat practice of the present invention by those skilled in the art usingconcentrations, amounts, contents, carbon numbers, and properties thatare outside of the range or different from a single value, will achievethe desired result, namely, effective treatment of a subject withvitamin B₁₂ deficiency which failed to respond to existing oral vitaminB₁₂ tablets as well as pharmaceutical compositions for such treatment.

EXAMPLE 1 Preparation of N-[8-(2-hydroxybenzoyl)amino]caprylic acid andSNAC

The preparation method for N-[8-(2-hydroxybenzoyl)amino]caprylic acidand SNAC involves the following steps: The starting material issalicylamide, which is converted to form Carsalam. The second stepinvolves the alkylation of Carsalam. The penultimate step is ahydrolysis to cleave the ethyl protection group at the end of the alkylchain and spring open the heterocyclic ring forming the free acid ofSNAC. In the final step, the sodium salt of the SNAC free acid is formedby reaction with a 1% excess stoichiometric amount of sodium hydroxidebase. Upon cooling the precipitated product is isolated bycentrifugation and vacuum dried prior to packaging. The in-processcontrols for the synthetic scheme are given in Table I.

TABLE I In-process controls for SNAC Manufacturing Process. In-ProcessStep Reaction Desired Product Specification Control 1 Carsalam Carsalam<10% HPLC salicylamide 2 Alkylation Alkylated <8% Carsalam HPLC Carsalam3 Hydrolysis SNAC Free  <0.5% LOD acid 4 Sodium Salt SNAC Sodium 95-105%HPLC salt

EXAMPLE 2 Preparation of Vitamin B₁₂ Tablets

The tablet die and punches are checked to ensure that they are clean andthat their surfaces are dusted with magnesium stearate powder. VitaminB₁₂, SNAC, carrier, excipient, emulsifier, stabilizer, sweetener,flavoring agent, diluent, coloring agent, solubilizing agent arescreened through a #35 sieve and transferred into a sealed containers.50 mg of Vitamin B₁₂ is weighed and mixed thoroughly with 11 grams of acarrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent,diluent, coloring agent and/or solubilizing agent. 100 vitamin B₁₂tablets are made, with each tablet containing 0.5 mg of Vitamin B₁₂ and110 mg of a carrier, excipient, emulsifier, stabilizer, sweetener,flavoring agent, diluent, coloring agent and/or solubilizing agent.These tablets are used as a control.

EXAMPLE 3 Preparation of Vitamin B₁₂ and SNAC Tablets

50 mg of Vitamin B₁₂, 1 gram of SNAC are weighed and thoroughly mixedwith 10 grams of a carrier, excipient, emulsifier, stabilizer,sweetener, flavoring agent, diluent, coloring agent and/or solubilizingagent. 100 vitamin B₁₂ tablets are made, with each tablet containing 0.5mg of Vitamin B₁₂. 10 mg of SNAC and 100 mg of a carrier, excipient,emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloringagent and/or solubilizing agent. The process is repeated to make tabletbatches containing 1.0 mg, 0.8 mg, 0.6 mg, 0.4 mg and 0.2 of VitaminB₁₂, respectively. These tablets have the following specifications forrelease of SNAC component:

Tests Specification Analytical Method Appearance White to light-tanpowder with AM001 pink hue Identification Test for Sodium Confirmspresence of Sodium USP <191> FTIR Conforms to reference standard USP<197K> Melting Range/Temperature 193-203° C. with a range not to USP<741> exceed 5° C. Water Content NMT 3.0% USP <921> Method I HeavyMetals <20 ppm USP <231> Method II Sodium Content 6.9 to 8.4% AM017Residual Solvents Ethanol Less than 4000 ppm AM008 Heptane Less than 500ppm AM008 Assay as SNAC Sodium salt 90.0-110.0% w/w AM016 (As Is)

EXAMPLE 4 Preparation of Tablets for Testing on Rats

Tablets with four types of different ingredients were made as follows:(1) 8.8 mg of vitamin B₁₂, 35 mg of SNAC were weighed, thoroughly mixedand made into a tablet for dosing on rat; (2) 8.8 mg of vitamin B₁₂, 35mg of SNAC and 5 mg of Capmul PG-8 were weighed, thoroughly mixed andmade into a tablet; (3) 8.8 mg of vitamin B₁₂, 35 mg of SNAC and 0.9 mgof Providone were weighed, thoroughly mixed and made into a tablet. Eachof the four processes was repeated to produce more tablets.

EXAMPLE 5 Dosing Sprague-Dawley Rats

Male Sprague-Dawley rats (325-350 g) were dosed with vitamin B₁₂intravenously (0.5 mg/kg) alone, or orally with the tablets made inExample 4 at a dose of 50 mg/kg vitamin B₁₂ alone or in combination withSNAC at 200 mg/kg. Blood samples were collected at 0, 3, 10, 20, 30, 60,120, 240 and 360 minutes post dosing. Plasma samples were analyzed forB12 by RIA. The model independent PK metrics obtained following B12-SNACcombination were compared to those obtained following B12 alone. Thetesting results are shown in Table 1.

TABLE 1 Comparative Testing Results for Vitamin B₁₂ Absorption AUC MeanCmax Tmax (ug * min/ Bio- Group (ug/mL) (min) mL) availability (N = 5)Mean S.D Mean S.D Mean S.D % 0.5 mg/kg 2.15 0.64 4.4 3.13 65.84 11Vitamin B₁₂ (IV) 50 mg/kg 0.14 0.07 52 17.9 28.72 13 0.42 Vitamin B₁₂alone (PO) 50 mg/kg 7.99 2.41 24 5.48 522.37 179 7.93 Vitamin B₁₂ + 200mg/kg SNAC (PO)

EXAMPLE 6 Preparation of Tablets for Testing on Human Subjects

Tablets were made from Cyanocobalamin, SNAC, Kollidon 90F, AnhydrousEmcompress USP/EP and Magnesium Stearate, NF/BP/EP/JP. Each tabletcontains the followings:

Ingredients mg/tablet Cyanocobalamin, USP (Intragranular) 5.00 SNAC(Intragranular) 100.00 Kollidon 90F, NF/EP/JP 2.00 (Providone K90;Intragranular) Anhydrous Emcompress USP/EP (Diabasic 70.00 CalciumPhosphate, Anhydrous; Intragranular) Anhydrous Emcompress USP/EP(Diabasic 21.00 Calcium Phosphate, Anhydrous; Extragranular) MagnesiumStearate, NF/BP/EP/JP 2.00 (extragranular) Total Weight 200.0

EXAMPLE 7 Dosing Human Subjects

Sixteen healthy male subjects were randomized to receive one of thefollowing treatments:

-   -   (1) Treatment B: a single oral dose of cyanocobalamin/SNAC (5 mg        cyanocobalamin/100 mg SNAC) administered in the fasted state as        a tablet. (6 subjects);    -   (2) Treatment C: a single oral dose of cyanocobalamin alone (5        mg cyanocobalamin, VitaLabs, commercial) administered in the        fasted state as a tablet. (6 subjects).    -   (3) Treatment D: a single intravenous dose of cyanocobalamin (1        mg cyanocobalamin) administered in the fasted state. (4        subjects). Each subject received a 1 mL intravenous injection of        a 1 mg/mL (1000 μg/mL) solution resulting in a total dose of 1        mg cyanocobalamin.

The subjects were fasted overnight prior to dosing and had no liquids(including water) consumption for at least one hour before and afterdosing. The oral forms of cyanocobalamin/SNAC tablets were administeredin a single dose as tablets with 50 mL of plain water. Twenty-five bloodsamples were drawn for cyanocobalamin analyses at the following timepoints: within 30 minutes pre-dose and at Minutes 2, 5, 10, 20, 30, 40,50, and at Hours 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 20and 24 post-dose.

Pharmacokinetic metrics was obtained following a model independentpharmacokinetic analysis of individual cyanocobalamin concentrations.Descriptive statistics was used to summarize the results.

Following 1 tablet of 5 mg B12/100 mg SNAC mean B12 peak concentrationis 12847±6613 pg/mL and occur within 1 hour post dose (mean tmax of0.50±0.21 hours). Mean AUClast (0-24) value is 54618±16392 hr*pg/mL. Thepercent coefficient of variation (% CV) is 51.5% for Cmax and 30.0% forAUC.

Following a single oral dose of cyanocobalamin alone (5 mgcyanocobalamin, VitaLabs, commercial) mean B12 peak concentration is1239±450 pg/mL and occur between 3 to 10 hours post-dose (mean tmax of6.8±3.2 hours). Mean AUClast (0-24) value is 23131±8343 hr*pg/mL. Thepercent coefficient of variation (% CV) is 36.3% for Cmax and 36.1% forAUC.

Following a single intravenous dose of cyanocobalamin (1 mgcyanocobalamin) administered in the fasted state (4 subjects). Mean B12peak concentration is 221287±80248 pg/mL and mean AUClast (0-24) valueis 215391±44602 hr*pg/mL. The percent coefficient of variation (% CV) is36.3% for Cmax and 20.7% for AUC.

The mean bioavailability of 1 tablet of 5 mg vitamin B12 alone, 1 tabletof 5 mg vitamin B12/100 mg SNAC, and 2 tablets of 5 mg vitamin B12/100mg SNAC are 2.15±0.77%, 5.07±1.52, and 5.92±3.05%, respectively. (Note:2 tablets of 5 mg vitamin B12/100 mg SNAC were dosed previously in apilot arm are designated Treatment A).

The mean tmax of 1 tablet of 5 mg vitamin B12 alone, 1 tablet of 5 mgvitamin B12/100 mg SNAC, and 2 tablets of 5 mg vitamin B12/100 mg SNACare 6.8±3.2 hours, 0.50±0.21 hours, and 0.54±0.32 hours, respectively.

No adverse events were observed during the given treatments. Allformulations appear to be safe and well tolerated.

It was found surprisingly that the extent of B12 absorption, measured asCmax and AUC, was significantly enhanced by the administration of thecyanocobalamin/SNAC combination. Vitamin B12 bioavailability was ˜240%greater for the 1 tablet of 5 mg B12/100 mg SNAC compared to 5 mg B12commercial formulation. Mean peak B12 concentrations following B12commercial oral formulation occurred significantly later compared tothat following the B12/SNAC combinations likely due to a different siteof absorption between the two oral formulations. This is consistent withliterature data describing intestinal absorption of B12 occurring in thedistal section of the gastrointestinal tract in the absence of thecarrier.

The principles, preferred embodiments, and modes of operation of thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein, however, is notto be construed as limited to the particular forms disclosed, sincethese are to be regarded as illustrative rather than restrictive.Variations and changes may be made by those skilled in the art, withoutdeparting from the spirit of the invention.

1. A method for administering vitamin B₁₂ to a subject to treat vitaminB₁₂ deficiency, comprising orally administering a pharmaceuticalcomposition comprising (1) from about 0.01 mg to about 25 mg of vitaminB₁₂ and (2) from about 1 mg to about 600 mg of at least one substanceselected from N-[8-(2-hydroxybenzoyl) amino]caprylic acid and itspharmaceutically acceptable salts to said subject.
 2. The method ofclaim 1, wherein said subject had failed to respond to existing oralvitamin B₁₂ treatment.
 3. The method of claim 1, wherein saidpharmaceutical composition is a tablet.
 4. The method of claim 1,wherein said pharmaceutical composition has a weight ratio of vitaminB₁₂ to monosodium N-[8-(2-hydroxybenzoyl) amino]caprylate of from about2:1 to about 1:700.
 5. The method of claim 4, wherein said weight ratiois from about 1:2 to about 1:600.
 6. The method of claim 4, wherein saidweight ratio is from about 1:3 to about 1:20.
 7. The method of claim 1,wherein said further comprises at least one of a carrier, excipient,emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloringagent, solubilizing agent or combinations thereof.
 8. An oralpharmaceutical composition comprising (1) from about 0.01 mg to about 25mg of vitamin B₁₂ and (2) from about 1 mg to about 600 mg of at leastone substance selected from N-[8(2-hydroxybenzoyl) amino]caprylic acidand its pharmaceutically acceptable salts.
 9. The pharmaceuticalcomposition of claim 8, wherein said pharmaceutical compositioncomprises a tablet.
 10. The pharmaceutical composition of claim 9,wherein said tablet comprises from about 0.01 to about 25 mg of vitaminB₁₂ and from about 50 to about 600 mg of monosodiumN-[8-(2-hydroxybenzoyl) amino]caprylate.
 11. The pharmaceuticalcomposition of claim 9, wherein said tablet comprises from about 1 toabout 15 mg of vitamin B₁₂ and from about 50 to about 200 mg ofmonosodium N-[8-(2-hydroxybenzoyl) amino]caprylate.
 12. Thepharmaceutical composition of claim 8, further comprising at least oneof a carrier, excipient, emulsifier, stabilizer, sweetener, flavoringagent, diluent, coloring agent, solubilizing agent or combinationsthereof.
 13. The method of claim 1, wherein said pharmaceuticalcomposition comprises about 0.1 to 1.5 mg of vitamin B₁₂ and from 25 to100 mg of monosodium N-[8-(2-hydroxybenzoyl) amino]caprylate.
 14. Themethod of claim 1, wherein the vitamin B₁₂ is a cobalamin.
 15. Themethod of claim 1, wherein the vitamin B₁₂ is cyanocobalamin.
 16. Themethod of claim 1, wherein the vitamin B₁₂ is hydroxocobalamin.
 17. Themethod of claim 1, wherein the vitamin B₁₂ is methylcobalamin.
 18. Themethod of claim 1, wherein the vitamin B₁₂ is 5-deoxyadenosylcobalamin.19. The method of claim 13, wherein the vitamin B₁₂ is a cobalamin. 20.The method of claim 13, wherein the vitamin B₁₂ is cyanocobalamin. 21.The method of claim 13, wherein the vitamin B₁₂ is hydroxocobalamin. 22.The method of claim 13, wherein the vitamin B₁₂ is methylcobalamin. 23.The method of claim 13, wherein the vitamin B₁₂ is5-deoxyadenosylcobalamin.
 24. The oral pharmaceutical composition ofclaim 8, wherein said pharmaceutical composition comprises about 0.1 to1.5 mg of vitamin B₁₂ and from 25 to 100 mg of monosodiumN-[8-(2-hydroxybenzoyl) amino]caprylate.
 25. The oral pharmaceuticalcomposition of claim 8, wherein the vitamin B₁₂ is a cobalamin.
 26. Theoral pharmaceutical composition of claim 8, wherein the vitamin B₁₂ iscyanocobalamin.
 27. The oral pharmaceutical composition of claim 8,wherein the vitamin B₁₂ is hydroxocobalamin.
 28. The oral pharmaceuticalcomposition of claim 8, wherein the vitamin B₁₂ is methylcobalamin. 29.The oral pharmaceutical composition of claim 8, wherein the vitamin B₁₂is 5-deoxyadenosylcobalamin.
 30. The oral pharmaceutical composition ofclaim 24, wherein the vitamin B₁₂ is a cobalamin.
 31. The oralpharmaceutical composition of claim 24, wherein the vitamin B₁₂ iscyanocobalamin.
 32. The oral pharmaceutical composition of claim 24,wherein the vitamin B₁₂ is hydroxocobalamin.
 33. The oral pharmaceuticalcomposition of claim 24, wherein the vitamin B₁₂ is methylcobalamin. 34.The oral pharmaceutical composition of claim 24, wherein the vitamin B₁₂is 5-deoxyadenosylcobalamin.
 35. The method of claim 1, wherein thesubstance in component (2) is monosodiumN-[8-(2-hydroxybenzoyl)amino]caprylate.
 36. The oral pharmaceutical ofclaim 8, wherein the substance in component (2) is monosodiumN-[8-(2-hydroxybenzoyl)amino]caprylate.